EP0981649A1 - Peptides favorisant le transport d'un agent actif dans les tissus, compositions les renfermant et leurs procedes d'utilisation - Google Patents

Peptides favorisant le transport d'un agent actif dans les tissus, compositions les renfermant et leurs procedes d'utilisation

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Publication number
EP0981649A1
EP0981649A1 EP98923466A EP98923466A EP0981649A1 EP 0981649 A1 EP0981649 A1 EP 0981649A1 EP 98923466 A EP98923466 A EP 98923466A EP 98923466 A EP98923466 A EP 98923466A EP 0981649 A1 EP0981649 A1 EP 0981649A1
Authority
EP
European Patent Office
Prior art keywords
peptide
transport
targeting
active agent
uptake
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP98923466A
Other languages
German (de)
English (en)
Other versions
EP0981649A4 (fr
Inventor
Daniel Joseph O'mahony
Vernon Leon Alvarez
Michela Apartment 89 SEVESO
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytogen Corp
Original Assignee
Elan Corp PLC
Cytogen Corp
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Filing date
Publication date
Application filed by Elan Corp PLC, Cytogen Corp filed Critical Elan Corp PLC
Publication of EP0981649A1 publication Critical patent/EP0981649A1/fr
Publication of EP0981649A4 publication Critical patent/EP0981649A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/70—Nanostructure
    • Y10S977/788—Of specified organic or carbon-based composition
    • Y10S977/802—Virus-based particle
    • Y10S977/803—Containing biological material in its interior
    • Y10S977/805—Containing drug

Definitions

  • the present invention relates to peptides which permit or facilitate the transport of drugs, macromolecules, or particles, such as drug-loaded nanoparticles, through human or animal tissues.
  • this invention relates to certain targeting peptides, their derivatives or peptidomimetics. and pharmaceutical compositions containing the same that are designed to target one or more transport or uptake pathways.
  • These targeting agents enhance the delivery and/or transport of an active agent through tissue, such as epithelial cells lining the lumenal side of the gastro-intestinal tract (GIT) when administered to a subject, especially when administered orally.
  • GIT gastro-intestinal tract
  • the epithelial cells lining the lumenal side of the GIT are a major barrier to drug delivery following oral administration.
  • transport pathways which can be exploited to facilitate drug delivery and transport: the transcellular. paracellular, carrier-mediated and transcytotic transport pathways.
  • the ability of a conventional drug, peptide, protein, macromolecule or nano- or microparticulate system to "interact" with one of these transport pathways may result in increased delivery of that drug or particle from the GIT to the underlying circulation.
  • folic acid which interacts with the folate receptor
  • mannose and cetylmannoside which interact with the mannose receptor
  • cobalamin which interacts with Intrinsic Factor.
  • leucine- and tyrosine-based peptide sorting motifs or internalization sequences exist, such as YSKV, FPHL, YRGV, YQTI, TEQF, TEVM, TSAF, YTRF, which facilitate uptake or targeting of proteins from the plasma membrane to endosomes.
  • Phage display libraries can be screened using specific membrane receptors or binding sites to identify peptides that bind specifically to the receptor or binding site.
  • the ability of certain motifs or domains of peptides or proteins to interact with specific membrane receptors, followed by cellular uptake of the protei receptor complex may point towards the potential application of such motifs in facilitating the delivery of drugs.
  • the identification of peptides or peptide motifs by their ability to interact with specific receptor sites or carrier sites, such as sites expressed on the apical side of the epithelial sites of the GIT, may not be able to determine, or may not be the most effective way to determine, the identity of peptides capable of enhancing the transport of an active agent, especially a drug- loaded nano- or microparticle, through tissues such as epithelial lining.
  • Non-receptor-based assays to discover particular ligands have also been used. For instance, a strategy for identifying peptides that alter cellular function by scanning whole cells with phage display libraries is disclosed in Fong et al., Drug Development Research 33:64-70 (1994). However, because whole cells, rather than intact tissue or polarized cell cultures, are used for screening phage display libraries, this procedure does not provide information regarding sequences whose primary function includes affecting transport across polarized cell layers. Additionally, Stevenson et al., Pharmaceutical Res. 12(9), S94 (1995) discloses the use of Caco-2 monolayers to screen a synthetic tripeptide combinatorial library for information relating to the permeability of di- and tri-peptides. While useful, this technique does not assess the ability of the disclosed di- and tri-peptides to enhance delivery of a drug, especially a drug-loaded nano-or microparticle formulation.
  • Co-pending PCT applications WO 97/17613 and WO 97/17614 disclose a method of identifying a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.
  • a predetermined amount of phage from a random phage library or preselected phage library is plated unto or brought into contact with a first side, preferably the apical side, of a tissue sample, either in vitro, in vivo or in situ, or polarized tissue cell culture.
  • the phage which is transported to a second side of the tissue opposite the first side, preferably the basolateral side is harvested to select transported phages.
  • the transported phages are amplified in a host and this cycle of events is repeated (using the transported phages produced in the most recent cycle) a predetermined number of times, such as from zero to six times, to obtain a selected phage library containing phage which can be transported from the first side to the second side.
  • the sequence of at least one random peptide coded by phage in the selected phage library is determined in order to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue.
  • Dynamin a 100 kDa member of the GTPase superfamily, is required for endocytosis at the plasma membrane in a large number of cell types, including neurones.
  • Dynamin was first identified as a brain protein localized in nerve terminals in association with clathrin-coated vesicles and a specific role of dynamin in synaptic vesicle recycling was proposed.
  • the recent studies of dynamin mutants in non- neuronal cells suggest a general role in receptor-mediated endocytosis.
  • Dynamin concentrates on clathrin-coated membrane surfaces and spontaneously self-assembles into helical arrays forming rings and stacks of interconnected rings at the necks of invaginated coated pits that become constricted. It appears that a concerted conformational change then closes the rings and pinches off the budding coated vesicles from the plasma membrane.
  • Dynamin is encoded by at least three different genes: dynamin I. which is predominantly expressed in neurons where it is concentrated in synaptic terminals; dvnamin II, which has a widespread tissue distribution and dynamin III, which is expressed primarily in the testis but also in lung and brain.
  • dynamin I and III expression has been shown to be upregulated throughout development of the brain whereas the levels of dynamin II mR A remain unchanged.
  • the three dynamin proteins encoded by these genes have a similar molecular weight of 94-100kDa, are approximately 70% identical to each other, and have a similar domain structure.
  • a GTP -binding domain in the amino-terminal region 300 amino acids with three consensus elements highly conserved among all GTPase superfamily members
  • a central pleckstrin homology (PH) domain a highly basic proline-rich carboxyl terminus
  • PH domain is a stretch of approximately 100 amino acids at the center of the dynamin molecule and is generally thought to be involved in protein-protein or protein-lipid interaction.
  • the C-terminal tail of Dynamin ( -100 amino acids) is characterized by a very high pi and is rich in proline residues (therefore termed PRD).
  • Proline is a unique amino acid due to its cyclic structure, which in polypeptide chains restricts the natural conformation of surrounding amino acids.
  • PRDs are therefore characteristically stiff structures that may protrude out from the protein to form sticky arms able to bind to a range of proteins.
  • the dynamin PRD region contains at least three domains which can interact with proteins containing src-homology (SH3) domains (for example, in amphiphysin, Grb2, isoforms of phospholipase C (PLC), PI3 kinase, among others).
  • SH3 proteins containing src-homology
  • SH3 -containing proteins are involved in signal transduction pathways mediating protein-protein and protein-cytoskeleton interactions. It has also been proposed that specific regions within the PRD mediate specific targeting of dynamin to clathrin-coated pits, indicating that the key targeting signal for dynamin is an SH3 protein.
  • the highly basic nature of the C-terminal domain of dynamin also enables its interaction with acidic phospholipids, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, endogenous brain vesicles and may, in part, explain the association of dynamin with plasma membranes. These ionic interactions seem to stimulate assembly of dynamin dimers, trimers, etc. and, as consequence of self- assembly, GTP hydrolysis is activated.
  • the PRD region can be phosphorylated by PKC in vitro and this phosphorylation stimulates intrinsic dynamin GTPase activity.
  • Intravenous drug administration suffers from numerous limitations, including (i) the risk of adverse effects resulting from rapid accumulation of high concentrations of drug, (ii) repeated injections which can cause patient discomfort; and (iii) the risk of infection at the site of repeated injections.
  • Subcutaneous injection is not generally suitable for delivering large volumes or irritating substances.
  • oral administration is generally more convenient, it is limited where the therapeutic agent is not efficiently absorbed by the gastrointestinal tract.
  • Site specific drug delivery or drug targeting can be achieved at different levels, including (i) primary targeting to a specific organ, (ii) secondary targeting to a specific cell type within that organ and (iii) tertiary targeting where the drug is delivered to specific intracellular structures (e.g., the nucleus for genes) (reviewed in Davis and Hum, 1994, In: Targeting of Drugs 4, (Eds), Gregoriadis, McCormack and Poste, 183-194).
  • DDS Drug Delivery Systems
  • Monoclonal antibodies are being used as both delivery vehicles targeted to tumor cells (reviewed by Pietersz, G.A.. 1990, Bioconjugate Chem. 1 : 89-95) and as imaging agents to carry molecules of drug or imaging agent to the tumor surface.
  • targeting agents and delivery systems that incorporate the targeting agents that are capable of enhancing the transport and/or uptake of an active agent though one or more tissue barriers.
  • peptide targeting agents, derivatives or peptidomimetics thereof that are effective in transporting drugs, including drug-loaded nano- and microparticles delivery systems, across a human or animal tissue barrier.
  • the present invention provides targeting agents that permit or facilitate transport of an active agent through a human or animal gastro-intestinal tissue.
  • These targeting agents are peptides or their derivatives (e.g., fragments) and peptidomimetics thereof, and the nucleotide sequences coding for the peptides and derivatives.
  • These targeting agents can be used to target an active agent to one or more transport and/or uptake pathways, such as endocytosis, carrier mediated uptake, transcytosis and pinocytosis.
  • the tissue through which transport and/or uptake is facilitated is of the duodenum, jejunum, ileum, ascending colon, transverse colon, descending colon, or pelvic colon.
  • the tissue is most preferably epithelial cells lining the lumenal side of the GIT.
  • the targeting agents of the present invention have use in facilitating transport of active agents from the lumenal side of the GIT into the systemic blood system, and/or in targeting active agents to the GIT.
  • the active agent can be targeted to receptor sites or transport pathways which are known to operate in the human gastrointestinal tract, thus facilitating its absorption into the systemic system.
  • the targeting agents of this invention have use in facilitating targeted drug delivery across other barrier systems in which the respective target receptor or transport and/or uptake pathway is found, for example, the blood brain barrier, the vasculature and the lung.
  • the targeting agents also have use in facilitating targeted drug delivery to tissues or cells in which the respective target receptor or uptake and/or transport pathway is found, including but not limited to the brain, heart, lung testis, spleen, liver, tumors, the eye and the like.
  • the transport and/or uptake mediating targeting agents disclosed in this invention can be used in combination with other targeting agents which bind to or target specific receptors.
  • the transport and/or uptake mediating targeting agents disclosed in this invention can be used in combination with agents that permit or allow mucoadhesion. In this manner, effective delivery of an active agent can advantageously occur via different mechnisms simultaneously thus providing enhanced bioavailability of the active agent.
  • the targeting agents can be used to coat the surface of nanoparticulate or microparticulate drug delivery vehicles. Such coatings can be performed by either direct adsorption of the targeting agent to the surface of the particulate system or alternatively by covalent coupling of the targeting agent to the surface of the particulate system, either directly or via a linking moiety or by covalent coupling of the targeting agent to the polymers used in the production of nanoparticulate or microparticulate drug delivery vehicles, followed by the utilization of such targeting agen polymer conjugates in the production of nanoparticulate or microparticulate drug delivery vehicles.
  • the invention also relates to derivatives and peptidomimetics of the peptides which are functionally active. / ' . e. , they are capable of displaying one or more known functional activities associated with the full-length peptide such as the ability to bind or compete with the full-length peptide for binding to epithelial cells.
  • the invention further relates to fragments of (and derivatives thereof) of the peptides which comprise one or more motifs of the targeting agent peptide.
  • Antibodies to the targeting agent peptides and targeting agent peptide derivatives are additionally provided. Such antibodies are useful to quantify the targeting agents to the drug delivery system, to quantify the uptake of the targeting agent or delivery system across a tissue barrier such as the GIT, and to quantify targeted drug delivery to target cells or tissues. Methods of production of the targeting agent peptides, derivatives, fragments and analogs, e.g., by recombinant means, are also provided.
  • the present invention also relates to therapeutic methods, pharmaceutical compositions and formulations based on the targeting agent peptides.
  • Formulations of the invention include but are not limited to targeting agent peptides or motifs and derivatives (including fragments) thereof; antibodies thereto; and nucleic acids encoding the targeting agent peptides or derivatives associated with an active agent.
  • the active agent is a drug or drug-containing nano- or microparticle.
  • the active agent is a drug or a nano- or microparticle.
  • the active agent is a drug encapsulated or drug loaded nano- or microparticle, such as a biodegradable nano- or microparticle, in which the peptide is physically adsorbed or coated or covalently bonded, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle.
  • the peptide can form the nano- or microparticle itself or can be directly conjugated to the active agent.
  • Such conjugations include fusion proteins in which a DNA sequence coding for the peptide is fused in-frame to the gene or cDNA coding for a therapeutic peptide or protein, such that the modified gene codes for a recombinant fusion protein in which the "targeting" peptide is fused to the therapeutic peptide or protein and where the "targeting" peptide increases the absorption of the fusion protein from the GIT.
  • viral vector systems can be modified to express the targeting agents on their surface such that the targeting agent is expressed as a fusion protein with a viral surface protein and, as such, permit the targeting of the virus to receptor systems or transport and/or uptake pathways found on target cells or tissues. Such modified viral vector systems can be used for gene delivery.
  • Fig. 1 shows the binding profiles of brain selected phage to Caco-2 fixed cells.
  • Fig. 1(A) Caco-2 P34, read at 30 min;
  • Fig. 1(B) Caco-2 P36, read at 20 min;
  • Fig. 1(C) Caco-2 P34, read at 10 min.
  • the neat sample corresponds to 10 10 phage/well;
  • Fig. 2 shows the uptake and transport of brain selected phage 41.1 and 1.4 across Caco-2 cell mono layers.
  • Fig. 2(A) shows the titer of phage eluted by acid treatment of cell monolayers after 2 hours incubation at 37°C;
  • Fig. 2(B) shows the titer of cell-associated phage detected in the cell lysate after 2 hours incubation at 37°C;
  • Fig. 2(C) shows the titer of phage from the basolateral medium after 6 hours incubation at 37°C of Caco-2 cells with phage suspension on the apical side;
  • Fig. 3 shows the first ten homologies for each of the eight targeting peptides expressed by phage transported from the rat duodenal lumen to the brain obtained from a Blast search against the non-redundant SwissProt database.
  • Figs. 4(A), 4(B) and 4(C) show selected homologies between peptides 41.1, 37.1 and 1.4 and known peptides/proteins, respectively;
  • Fig. 5 shows the evaluation of the synthetic peptides dynamin 41.2 and peptide 41.1 in ELISA assays for binding to membrane PI 00 (Fig. 5 A) and cytosolic SI 00 (Fig. 5B) fractions isolated from Caco-2 cells.
  • the samples consisted of the following dansylated peptides: (1) negative control scrambled PAX2-coated nanoparticles, (2) positive control HAX42-coated nanoparticles, (3) 41.1 -coated particles, and (4) 41.2-coated particles.
  • the subcellular fractions P100 and S100 were used at a concentration of 50 ⁇ g/ml and the background has been subtracted;
  • Fig. 6 shows the binding of particles that are coated with dansylated targeting peptides and particles that are not coated to PI 00 (Fig. 6N) and SI 00 (Fig. 6B) Caco-2 fractions.
  • the subcellular fractions PI 00 and SI 00 were used at a concentration of 10, 5, 1, and 0.1 ⁇ g/well whereas the coated particles were used at 62.5 ⁇ g/well. The background has not been subtracted.
  • Fig. 7 shows the insulin plasma levels following intestinal administration of negative control (PBS); insulin particles (no peptide; 300 iu); positive control insulin HAX42-particles (40 mer; 300iu); and study group particles insulin peptide 41.1 particles (labeled Clone 41.1 particles; 43 mer; 300 iu) and insulin dynamin 41.2 particles (43 mer; 300 iu) according to this invention.
  • the particles were administered in a rat duodenal open loop model.
  • This invention discloses specific targeting agents comprising targeting peptides and their derivatives and peptidomimetics thereof that are capable of facilitating the delivery or transport of an active agent such as a drug across human or animal tissues, including without limitation GIT epithelial layers, alveolar cells, endothelial cells of the blood-brain barrier, vascular smooth muscle cells, vascular endothelial cells, renal epithelial cells, M cells of the Peyers Patch, and hepatocytes.
  • delivery systems e.g., nanoparticles, microparticles, liposomes, micelles, and viral vector systems could be coated externally with, be linked to or be comprised of these "homing" peptides to permit targeted delivery of encapsulated drugs across particular tissues.
  • fusion proteins can be synthesized, either in vivo or in vitro, whereby the peptide is fused in-frame to a therapeutic peptide or protein active agent such that the peptide enhances the delivery or transport of the therapeutic peptide or protein across the tissue.
  • human or animal tissue includes, without limitation, the duodenum, jejunum, ileum, ascending colon, transverse colon, descending colon, pelvic colon, the vascular endothelium which line the vascular system, the vascular endothelial cells which form the blood brain barrier, vascular smooth muscle, alveolar, liver, kidney, bone marrow, heart, spleen, pancreas, thymus, brain, spinal, neuronal and retinal eye tissue.
  • human or animal tissue refers to animal tissue explicitly including human tissue.
  • the term "active agent” includes, without limitation, imaging agents or any drug or antigen or any drug- or antigen-loaded or drug- or antigen- encapsulated nanoparticle, microparticle, liposome, viral vector system or micellar formulation capable of eliciting a biological response in a human or animal.
  • drug- or antigen-loaded or drug- or antigen-encapsulated formulations include those in which the active agent is encapsulated or loaded into nano- or microparticles, such as biodegradable nano- or microparticles, and which have the peptide adsorbed, coated or covalently bonded, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle.
  • the peptide can form the nano- or microparticle itself or the peptide can be covalently attached to the polymer or polymers used in the production of the biodegradable nano- or microparticles or drug-loaded or drug-encapsulated nano- or microparticles or the peptide can be directly conjugated to the active agent.
  • conjugations to active agents include fusion proteins in which a DNA sequence coding for the peptide is fused in- frame to the gene or cDNA coding for a therapeutic peptide or protein such that the modified gene codes for a recombinant fusion protein.
  • drug includes, without limitation, any pharmaceutically active agent.
  • Representative drugs include, but are not limited to, peptides or proteins, hormones, analgesics, anti-migraine agents, anti-coagulant agents, anti-emetic agents, cardiovascular agents, anti-hypertensive agents, narcotic antagonists, chelating agents, anti-anginal agents, chemotherapy agents, sedatives, anti-neoplasties, prostaglandins, and antidiuretic agents.
  • Typical drugs include peptides, proteins or hormones such as insulin, calcitonin, calcitonin gene regulating protein, atrial natriuretic protein, colony stimulating factor, betaseron, erythropoietin (EPO), interferons such as ⁇ , ⁇ or ⁇ interferon, somatropin, somatotropin, somatostatin, insulin-like growth factor (somatomedins), luteinizing hormone releasing hormone (LHRH), tissue plasminogen activator (TPA), growth hormone releasing hormone (GHRH), oxytocin, estradiol, growth hormones, leuprolide acetate, factor VIII, interleukins such as interleukin-2, and analogs thereof; analgesics such as fentanyl, sufentanil, butorphanol, buprenorphine.
  • interferons such as ⁇ , ⁇ or ⁇ interferon
  • somatropin somatotropin,
  • levorphanol levorphanol, morphine, hydromorphone, hydocodone, oxymorphone, methadone, lidocaine, bupivacaine, diclofenac, naproxen, paverin, and analogs thereof; anti-migraine agents such as heparin, hirudin, and analogs thereof; anti-coagulant agents such as scopolamine, ondansetron, domperidone, etoclopramide, and analogs thereof; cardiovascular agents, anti-hypertensive agents and vasodilators such as diltiazem, clonidine, nifedipine, verapamil, isosorbide-5-mononitrate, organic nitrates, agents used in treatment of heart disorders and analogs thereof; sedatives such as benzodiazeines, phenothiozines and analogs thereof; narcotic antagonists such as naltrexone, naloxone and analogs thereof; chel
  • Representative drugs also include but are not limited to antisense oligonucleotides, genes, gene correcting hybrid oligonucleotides, ribozymes, aptameric oligonucleotides, triple-helix forming oligonucleotides, inhibitors of signal transduction pathways, tyrosine kinase inhibitors and DNA modifying agents.
  • Drugs that can be used also include, without limitation, systems containing gene therapeutics, including viral systems for therapeutic gene delivery such as adenovirus, adeno-associated virus, retroviruses, herpes simplex virus, Sindbus virus, liposomes, cationic lipids, dendrimers, and enzymes.
  • gene delivery viruses can be modified such that they express the targeting peptide on the surface so as to permit targeted gene delivery.
  • Targeting agent peptides, derivatives and peptidomimetics The invention relates to targeting agent peptides and derivatives (including but not limited to fragments) and peptidomimetics thereof.
  • targeting agent peptides include but are not limited to those containing as primary amino acid sequences, all or part of the amino acid sequences substantially as depicted in Tables 4 and 7.
  • Derivatives of the targeting agent peptides include peptides in which certain amino acid residues are replace or substituted by other amino acid residues of similar properties.
  • an acidic residue could be replace by another acidic residue
  • a hydrophobic residue could be replace by another hydrophobic residue
  • a non-charged residue could be replaced by another non-charged residue.
  • any residue or residues of the targeting agent peptides can be replace by other natural or non-natural residues so as to either increase affinity or specificity for a target receptor or transport uptake pathway or, alternatively, to broaden binding or interaction with other related receptors or transport pathways, to increase stability in vivo to protease digestion or other chemical or molecules detrimental to the targeting agent peptide.
  • targeting agent peptides may contain multiple repeats comprising all or a fragment of a targeting agent peptide, in particular a fragment of the same comprising the targeting domain or that domain which facilitates drug absorption in vivo or in vitro.
  • a combination of targeting agents are incorporated into the drug delivery system, preferably having at least one targeting agent component which targets a specific receptor site while the other targeting agent component targets an endogenous transport and/or uptake pathway in a target cell or tissue.
  • the active agent could be modified to target a receptor in the GIT together with one or more targeting agents identified in this invention, which target transport and/or uptake pathways.
  • nucleic acids encoding such peptides, derivatives and peptide analogs are also provided. Furthermore, in view of the degeneracy of the genetic code, the nucleic acid sequence can be modified by substitution of one codon with another codon without changing the identity of the targeting agent peptide coded for by the resulting nucleic acid sequence or nucleic acid sequences. Related nucleic acid sequences are envisaged which code for derivatives of the targeting agent peptide, including those in which certain amino acid residues are replace or substituted by other amino acid residues of similar properties.
  • nucleic acid sequences are envisaged which code for targeting agent peptides in which an acidic residue could be replaced by another acidic residue, a hydrophobic residue could be replace by another hydrophobic residue, a non-charged residue could be replaced by another non-charged residue.
  • nucleic acid sequences are envisaged which code for targeting agent peptides in which any residue or residues of the targeting agent peptides can be replaced by other natural residues so as to either increase affinity or specificity of the resulting targeting agent ligands for a target receptor or transport pathway, or alternatively, to broaden binding or interaction with other related receptors or transport pathway, to increase stability in vivo to protease digestion or other chemicals or molecules detrimental to the targeting agent peptide.
  • nucleic acid sequences may code for proteins that contain multiple repeats comprising all or a fragment of a targeting agent peptide, in particular, a fragment or fragments of the same comprising the targeting domain or the domain that facilitates drug absorption in vivo or in vitro.
  • Targeting agent peptides whose amino acid sequence comprise, or alternatively, consists of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, and 18 or a portion thereof that facilitates the delivery and/or transport of an active agent across human or animal tissues are provided.
  • the derivative or peptidomimetic is functionally active, i.e., capable of exhibiting one or more functional activities associated with a full-length targeting agent peptides.
  • such derivatives or peptidomimetics which have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for immunization.
  • the derivatives or analogs have the ability to bind to Caco-2 cells, in vitro, or to intestinal tissue, in vivo, or to other non-epithelial cells or tissues in vivo or in vitro.
  • peptidomimetics may contain multiple repeats comprising all or a fragment of a targeting agent peptide or peptidomimetic, particularly a fragment comprising the targeting domain or domains which facilitate drug absorption in vivo or in vitro.
  • derivatives of the targeting agent peptide sequences can be made by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other nucleotide sequences which encode substantially the same amino acid sequence may be used in the practice of the present invention. These include but are not limited to nucleotide sequences which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change.
  • the targeting agent peptide derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a targeting agent peptide including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • peptides 10, 15, 20, 25, 30 or 35 (contiguous) amino acids of the full-length targeting agent peptide are provided.
  • such peptides are not more than 20, 30, 40, 50, or 75 amino acids in length.
  • peptides may contain multiple repeats comprising all or a fragment of a targeting agent peptide, particularly a fragment comprising the targeting domain or that domain or domains which facilitate drug absorption in vivo or in vitro.
  • Targeting agent peptides include but are not limited to those molecules comprising regions that are substantially homologous to targeting agent peptides or fragments thereof (e.g., at least 50%, 60%, 70%, 80% or 90% identity) (e.g., over an identical size sequence or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art) or whose encoding nucleic acid is capable of hybridizing to a coding targeting agent peptide sequence, under stringent, moderately stringent, or nonstringent conditions.
  • the targeting agent peptides, derivatives and peptidomimetics of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned targeting agent peptide gene sequence can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual. 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).
  • the sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the targeting agent peptides-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutagenesis Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C, et al, 1978, J. Biol. Chem 253:6551), use of TAB® linkers (Pharmacia), use of PCR primers containing mutation(s) for use in amplification, etc.
  • Targets of the targeting agent peptide sequence may also be made at the protein level. Included within the scope of the invention are targeting agent peptide fragments or other derivatives or analogs which are differentially modified during or after translation or chemical synthesis, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin. chymotrypsin. papain. V8 protease, NaBH 4 ; acetylation, formylation. oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc. In a specific embodiment, the amino- and/or carboxy-termini are modified.
  • targeting agent peptides and derivatives thereof can be chemically synthesized.
  • a peptide corresponding to all or a portion of a GIT transport receptor-binding peptide or multiple repeats of a domain or domains within the targeting agent peptide which comprises the desired domain or which mediates the desired activity in vitro can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the GIT transport receptor- binding peptide sequence.
  • Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, ⁇ -Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, C ⁇ -methyl amino acids, N ⁇ -methyl amino acids, and amino acid analogs in general.
  • the amino acid can be D (dextrorotary) or L (levorotary).
  • the targeting agent peptide derivative is a chimeric, or fusion, peptide comprising a targeting agent peptide or fragment thereof or multiple repeats thereof (preferably consisting of at least a domain or motif of the targeting agent peptide, or at least 6, 10, 15, 20, 25, 30 or all amino acids of the targeting agent peptides or a binding portion thereof) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different peptide.
  • such a chimeric peptide is produced by recombinant expression of a nucleic acid encoding the protein (comprising a transport receptor-coding sequence joined in- frame to a coding sequence for a different protein).
  • a nucleic acid encoding the protein comprising a transport receptor-coding sequence joined in- frame to a coding sequence for a different protein.
  • Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art.
  • such a chimeric product may be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
  • Chimeric genes comprising portions of targeting agent nucleic acid fused to any heterologous protein-encoding sequences may be constructed.
  • a specific embodiment relates to a chimeric protein comprising a fragment targeting agent peptides of at least six amino acids.
  • the targeting agent peptides and derivatives thereof of the invention can be assayed for binding activity by suitable in vivo or in vitro assays, e.g., as described in the examples infra and/or as will be known to the skilled artisan.
  • the invention relates to targeting agent peptide derivatives, in particular targeting agent peptide fragments and derivatives of such fragments, that comprise, or alternatively consist of, one or more domains of a targeting agent peptide.
  • targeting agent peptide derivatives in particular targeting agent peptide fragments and derivatives of such fragments, that comprise, or alternatively consist of, one or more domains of a targeting agent peptide.
  • examples of such domains are identified in the examples infra.
  • the peptides and derivatives of the present invention may be chemically synthesized or synthesized using recombinant DNA techniques.
  • peptides may be prepared chemically by methods that are known in the art.
  • solid phase peptide synthesis consists of coupling the carboxyl group of the C-terminal amino acid to a resin and successively adding N-alpha protected amino acids.
  • the protecting groups may be any known in the art. Before each new amino acid is added to the growing chain, the protecting group of the previous amino acid added to the chain is removed.
  • the coupling of amino acids to appropriate resins is described by Rivier et al., U.S. Patent No. 4,244,946.
  • Such solid phase syntheses have been described, for example, by Merrifield, 1964, J. Am. Chem. Soc.
  • Purification of the synthesized peptides can be carried out by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography, high performance liquid chromatography (HPLC)), centrifugation, differential solubility, or by any other standard technique.
  • chromatography e.g., ion exchange, affinity, and sizing column chromatography, high performance liquid chromatography (HPLC)
  • HPLC high performance liquid chromatography
  • Biological peptide libraries can be used to express and identify peptides that bind to GIT transport receptors. According to this second approach, involving recombinant DNA techniques, peptides can by way of example be expressed in biological systems as either soluble fusion proteins or viral capsid proteins.
  • a targeting peptide, its fragments or other derivatives, or analogs thereof may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • Various procedures known in the art may be used for the production of polyclonal antibodies to a targeting peptide or derivative or analog.
  • various host animals can be immunized by injection with the native targeting peptides, or a synthetic version, or derivative (e.g., fragment) thereof, including but not limited to rabbits, mice, rats, etc.
  • adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B- cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies Colde et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545).
  • human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).
  • techniques developed for the production of "chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay).
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies specific to a domain of a targeting peptide are also provided.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the targeting peptide sequences of the invention, e.g., for imaging these peptides after in vivo administration (e.g., to monitor treatment efficacy), measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc.
  • antibodies or antibody fragments specific to a domain of a targeting peptide or to a derivative of a peptide, such as a dansyl group or some other epitope introduced into the peptide can be used to 1) identify the presence of the peptide on a nanoparticle or other substrate; 2) quantify the amount of peptide on the nanoparticle; 3)measure the level of the peptide in appropriate physiological samples; 4) perform immunohistology on tissue samples; 5) image the peptide after in vivo administration; 6) purify the peptide from a mixture using an immunoaffinity column or 7) bind or fix the peptide to the surface of a nanoparticle.
  • compositions comprising the targeting agent peptides, derivatives and peptidomimetic of the invention bound to a material comprising an active agent.
  • Such compositions have use in targeting the active agent to the GIT and/or in facilitating transfer through the lumen of the GIT into the systemic circulation.
  • the active agent is an imaging agent
  • such compositions can be administered in vivo to image the GIT (or particular transport receptors thereof).
  • a active agent is therapeutically or prophylactically administered to a human patient.
  • the invention provides for treatment of various diseases and disorders by administration of a therapeutic compound (termed herein "Therapeutic”).
  • a therapeutic compound include but are not limited to: targeting peptides, and derivatives (including fragments) and peptidomimetics thereof that are capable of facilitating the delivery or transport of an active agent across human or animal tissues, bound to an active agent of value in the treatment or prevention of a disease or disorder (preferably a mammalian, most preferably human, disease or disorder).
  • Therapeutics also include but are not limited to nucleic acids encoding the targeting agent peptides, derivatives or peptidomimetics bound to such a therapeutic or prophylactic active agent.
  • the active agent is preferably a drug.
  • the invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a Therapeutic of the invention.
  • the Therapeutic is substantially purified.
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
  • any disease or disorder of interest amenable to therapy or prophylaxis by providing a drug in vivo systemically or by targeting a drug in vivo to the GIT (by linkage to a targeting agent peptide, derivative or peptidomimetic of the invention) can be treated or prevented by administration of a Therapeutic of the invention.
  • diseases may include but are not limited to hypertension, diabetes, osteoporosis, hemophilia, anemia, cancer, migraine, and angina pectoris, to name but a few.
  • any route of administration known in the art may be used, including but not limited to oral, nasal, topical, intravenous, intraperitoneal, intradermal, mucosal, intrathecal, intramuscular, etc.
  • administration is oral; in such an embodiment the targeting agent peptide, derivative or peptidomimetic of the invention acts advantageously to facilitate transport of the therapeutic active agent through the lumen of the GIT into the systemic circulation.
  • a targeting agent peptide or motif of interest is associated with a therapeutically or prophylactically active agent, preferably a drug or drug-containing nano- or microparticle.
  • the active agent is a drug encapsulating or drug loaded nano- or microparticle, such as a biodegradable nano- or microparticle, in which the peptide is physically adsorbed or coated or covalently bonded, such as directly linked or linked via a linking moiety, onto the surface of the nano- or microparticle.
  • the peptide can form the nano- or microparticle itself or can be directly conjugated to the active agent.
  • Such conjugations include fusion proteins in which a DNA sequence coding for the peptide is fused in-frame to the gene or cDNA coding for a therapeutic peptide or protein, such that the modified gene codes for a recombinant fusion protein in which the "targeting" peptide is fused to the therapeutic peptide or protein and where the "targeting" peptide increases the absorption of the fusion protein from the GIT.
  • the particles range in size from 200-600 nm.
  • a targeting agent peptide is bound to a slow- release (controlled release) device containing a drug.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al, Science 228:190 (1985); During et al., Ann. Neural. 25:351 (1989); Howard et al., J.
  • compositions comprise a therapeutically effective amount of a Therapeutic, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the Therapeutics of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
  • the amount of the Therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • the NH 2 -terminal amino acid sequence of the absorption proteins pill and pVIII coded by Escherichia coli filementous bacteriophage phage such as fd can be modified by recombinant DNA technology to include a library of random peptide sequences of defined length (Cwirla et al., Proc. Natl. Acad. Sci. USA 87:6378-6382 (1990)).
  • a DNA library of modified phage fd sequences, coding for variable pill or pVIII proteins can be constructed and propagated in E. coli.
  • Phage display libraries such as these can be used in a random screening approach or a preselected phage library or subpopulation from a phage display library in a preselected screening approach in order to determine the identity of peptide sequences which enhance the delivery of the bacteriophage from either the apical to basolateral side or the basolateral to apical side of either cultured model systems or in in vitro, in situ or in vivo tissue samples.
  • Peptides that enhance the delivery from the apical to basolateral side e.g., gut side to blood side
  • plating on the basolateral side might determine peptides useful for raising a mucosal immune response to an antigen administered IV, subcutaneously, transdermally or by the opthalmic route.
  • the peptides coded by triple (or multiple) DNA inserts have the capacity to code for longer and/or more diverse peptides. Such longer peptides have a greater capacity to adopt secondary and tertiary structures as opposed to shorter peptides, such as a 15-mer peptide.
  • This capacity of peptides to adopt defined secondary and/or tertiary structures coded by those phages containing multiple or triple DNA inserts may in-turn account for the selection of these types of phages from random phage display libraries during selection or panning procedures.
  • transport mechanisms operate in epithelial cells. Some transport mechanisms are carrier mediated, whereby a carrier or receptor will bind to a ligand and transport the bound ligand into or through the epithelial cell. Other transport systems operate by transcytosis, whereby a carrier or receptor site will bind a ligand. the carrier: ligand complex is internalized by endocytosis and thus delivers a ligand (or drug) into or through the cell.
  • peptides disclosed by this invention were discovered by use of an in vivo random screening approach that identified peptide sequences that interacted with undefined or unknown receptor/carrier sites in tissues, such as epithelial cells.
  • This screening approach included contacting a predetermined amount of phage from a random phage library or a preselected phage library with a first side of a tissue barrier in vivo (such as injecting phage into the lumen of an animal), harvesting phage which is transported to the opposite side of the tissue barrier to select transported phage (such as harvesting phage from brain tissue), amplifying the transported phage in a host and identifying at least one random peptide coded by a transported phage to identify a peptide which permits or facilitates the transport of an active agent through a human or animal tissue. If desired, the contacting, harvesting and amplifying steps can be repeated a predetermined number of times using the transported phage obtained in the previous cycle.
  • the corresponding peptide sequences coded by the selected phages obtained by the procedures above and identified following DNA sequencing of the appropriate gene III or gene VIII genes of the phage, were synthesized.
  • the binding and transport of the synthetic peptide itself across the model cell culture or isolated tissue system (such as colonic) permits direct assessment of the transport characteristics of each individual peptide.
  • fusion of the selected peptide(s) sequences with other peptides or proteins permits direct assessment of the transport of such chimeric proteins or peptides across the model systems.
  • Such chimeric proteins or peptides can be synthesized either in vitro or by conventional recombinant technology techniques whereby the cDNA coding for the transporting peptide and the cDNA coding for the drug peptide or protein are ligated together in- frame and are cloned into an expression vector which in turn will permit expression in the desired host, be it prokaryotic cells or eukaryotic cells or transgenic animals or transgenic plants.
  • the cDNAs coding for the modified NH 2 -terminal region of the pill proteins can be subcloned into the genes or cDNAs coding for selected protein molecules (e.g., calcitonin, insulin, interferons, interleukines, cytokines.
  • EPO, colony stimulating factors etc. and these modified genes or cDNAs can be expressed in E. coli or suitable mammalian cells or transgenic animals or transgenic plants.
  • the expressed recombinant proteins can be purified and their transcellular, carrier-mediated, transcytotic and/or paracellular transport across human or animal tissue can be verified.
  • the transporting peptides can be used to coat the surface of nanoparticulate or microparticulate drug delivery vehicles.
  • Such coatings can be performed by either direct adsorption of the peptide to the surface of the particulate system or alternatively by covalent coupling of the peptide to the surface of the particulate system, either directly or via a linking moiety or by covalent coupling of the peptide to the polymers used in the production of nanoparticulate or microparticulate drug delivery vehicles, followed by the utilization of such peptide :polymer conjugates in the production of nanoparticulate or microparticulate drug delivery vehicles.
  • D38 and DC43 are random phage display libraries in which gene III codes for random peptides of 38 and 43 residues in size, respectively. These libraries are described in McConnell et al, Molecular Diversity 1:154-176 (1995), US Serial No. 310,192 filed September 21, 1994, US Serial No. 488,161 filed June 7, 1995, and WO 96/09411, which references are hereby incorporated by reference.
  • a large scale preparation of each of the bacteriophage libraries was made in the E. coli host strain K91Kan.
  • a single K91Kan colony was innoculated into a sterile 50 ml tube containing 20 ml LB broth (Yeast extract (Gibco) - 1 g; Tryptone (Gibco) - 2 g; NaCl - 1 g; and distilled water - 200 ml) together with kanomycin (final concentration lOO ⁇ g/ml) and grown to mid log phase with 200 rpm agitation at 37° C (OD 0.45 at 600 nm).
  • the cells were allowed to incubate with gentle shaking (100 rpm, 37° C) for 5 min to regenerate sheared F pili.
  • the cells were pelleted by centrifugation at 2200 rpm for 10 min at room temperature, the supernatant removed and the cells gently resuspended in 20 ml 80 mM NaCl and shaken gently (100 rpm, 37° C) for 45 min.
  • the cells were centrifuged again and the cell pellet was gently resuspended in 1 ml cold NAP buffer (NaCl (5 M stock) - 1.6 ml; NH 4 H 2 PO 4 (0.5 M stock, pH 7.0) - 10 ml; and distilled water - 88.4 ml).
  • the cells were stored at 4°C and remained infectable for 3-5 days.
  • the primary libraries were amplified by inoculating two 1 1 flasks containing 100 ml terrific broth with 1 ml of an overnight culture of K91Kan cells (grown in LB + 100 ⁇ g/ml kanamycin). This culture was incubated at 37°C and 200 rpm until the OD 600 of a 1 : 10 dilution was 0.2 and then further incubated for 5 min at 37°C and 200 rpm to allow sheared F pili to regenerate. 10 ⁇ l of the primary library was added to each flask with continued slow shaking for 15 min.
  • Each culture was poured into a prewarmed 2 1 flask containing 1 1 LB + 0.22 ⁇ g/ml tetracycline and shaken at 200 rpm for 35 min. 1 ml of 20 mg/ml tetracycline was added and 7 ⁇ l samples were removed from each flask. The flasks were replaced in an incubator with continued shaking overnight. 200 ⁇ l of various serial dilutions (10 "4 ,10 "6 , 10 "8 , 10 "10 dilutions) of each culture were spread on LB + 40 ⁇ g/ml tetracycline and 100 ⁇ g/ml kanamycin plates and incubated overnight. The colonies were counted.
  • Phage band - upper band approximately 5 mm, faint, blue, non-flocculent PEG - lower band, narrow, stringy, flocculent. opaque, white
  • the fluid was aspirated off to 2 mm above the phage band and the phage band was withdrawn using a sterile wide aperture transfer pipette and placed in a 26 ml polycarbonate centrifuge tube.
  • the tube was filled to the shoulder with TBS, mixed and centrifuged at 50,000 rpm for 4 h at 4°C in the 60Ti rotor (repeated).
  • the pellet was dissolved in 10 ml TBS by gentle vortexing and allowed to soften overnight in the cold and revortexed (repeated).
  • the pellet was then dissolved in TBS (2 ml per litre of original culture) by vortexing, allowed to soften overnight at 4°C and revortexed.
  • the tube was centrifuged briefly to drive solution to the bottom of the tube and transferred to 1.5 ml microtubes.
  • Sodium azide (0.02%) can be added and the solution can be heated to 70°C for 20 min to kill residual microorganisms. Following microfuging for 1 min to clear the solution, the supernatant was transferred to sterile microtubes and stored at 4°C.
  • 200 ⁇ l of a 1 :100 dilution was scanned from 240-320 nm to determine the concentration of physical particles and titre 10 ⁇ l of a 10 "8 dilution on 10 ⁇ l of starved K91Kan cells.
  • 200 ⁇ l of the infections was spread on LB (+ 40 ⁇ g/ml tetracycline and 100 ⁇ g/ml kanamycin) plates, incubated at 37 °C for 24 h and counted the number of colonies to determine the titre of infectious units in the phage stocks.
  • the Caco-2 (ATCC designation: CCL 248; derived from a lung metastasis of a colon carcinoma in a 72-year old male) were cultured initially in 25cm 2 flasks, until they reached confluency. T84 cells were grown in 1 :1 DMEM:Ham " s F12 medium containing 2mM glutamine, 15mM HEPES, 10% fetal calf serum (FCS), 1 % MEM non essential amino acids and 50U ml "1 penicillin and 50 ⁇ g ml "1 streptomycin.
  • Caco-2 cells were grown in DMEM + glutamax-1 containing 10% FCS, 1% MEM non essential amino acids, 50U ml "1 penicillin and 50 ⁇ g ml '1 streptomycin. All cells were incubated at 37°C in 95% O 2 / 5% CO 2 . At confluence the cells were used to seed snapwells.
  • the seeding of snapwells was essentially as follows for T-84 cells (a concentration of 1 x 10 6 cells/1.0ml of medium is required for each 12mm snapwell; a 100% confluent flask of T84 contains approximately 8 x 10 ⁇ cells and would be sufficient to seed 8 snapwells).
  • the flasks were trypsinised and cells were carefully resuspended. making sure there are no clumps or air bubbles.
  • 2.6ml of tissue culture medium is placed in the bottom of the wells and 0.1ml on the filter and placed in the incubator for 10 mins at 37°C. 1.5ml of the cell suspension was added to each filter, being careful not to let any fall into the bottom of the well. The filter was placed back in the incubator and checked after 24 hrs.
  • the cells were routinely monitored for adequate TER using an EVOM chopstick epithelial voltometer (WPI).
  • WPI EVOM chopstick epithelial voltometer
  • the seeding of Caco-2 cells was essentially the same as for T-84 cells except that they are seeded at 5 x 10-> rather than 1 10° cells/ snapwell.
  • the subsequent maintenance and feeding of the cells on the snapwells was as follows: when feeding the wells, the medium was removed from the basolateral side of the snapwell first. The medium was removed from the monolayer with a pipette being careful not to touch the filter and then 1ml of growth medium was place onto the apical side and 2ml of growth medium into the basolateral side. Spillages of medium on the sides of the plate outside the well were checked for and swabbed with a cotton bud moistened with alcohol if necessary. Following seeding on the snap wells, the cells were fed on a daily basis and were cultured on the snapwells for between 21-30 days, during which time the cells spontaneously differentiated and become polarized.
  • Enzyme linked immuno-sorbent assay for fd-derived phage on Caco-2 cells Caco-2 cells (lOO ⁇ l) were grown to confluence in 96 well tissue culture plates
  • Each well was filled with 200 ⁇ l of 0.1% phenylhydrazine-DPBS (0.1% phenylhydrazine in DPBS) and incubated for 1 h at 37°C. Subsequently, the contents of the microtitre wells were emptied by inversion/flicking and the wells were washed three times with DPBS. 200 ⁇ l of 0.5% BSA in DPBS was added to each well followed by incubation for 1 h at room temperature. Each well was next washed three times in 1% BPT (1% BSA, 0.05% Tween 20 in DPBS).
  • Phage samples 100 ⁇ l in 1% BPT (either neat phage at 10 10 pfu/ml or 1 :25 or 1 :100 dilutions thereof) were added to the wells, followed by incubation at room temperature for 2 h. The contents of the microtitre wells were removed by inversion flicking and the wells were washed five times in 1% BPT.
  • HRP horseradish peroxidase
  • Anti-M13 conjugate HRP/anti-M13 conjugate:horseradish peroxidase conjugated to sheep anti-M13 IgG; 1 :5000 working dilution in 1% BPT; Pharmacia 27-9402-01
  • HRP/anti-M13 conjugate:horseradish peroxidase conjugated to sheep anti-M13 IgG 1 :5000 working dilution in 1% BPT; Pharmacia 27-9402-01
  • the contents of the microtitre wells were again removed by inversion/flicking and the wells were washed five times in 1% BPT.
  • TMB substrate solution (3,3',5',5-tetramethylbenzidine; Microwell Peroxidase Substrate System; Kirkegaard & Perry Laboratories CN 50-76-00; prepared by mixing equal amounts of TMB Peroxidase Substrate A and Peroxidase Solution B in a glass container immediately before use) was added to each well, followed by incubation at room temperature for 20-60 min. Thereafter, absorbance readings were read at 650 nm on a microtitre plate reader.
  • Harvesting site tissue such as brain, heart, kidney, spleen, liver, pancreas. duodenum or ileum tissue, is collected from animals following introduction in vivo of the phage display library at a site separated from the harvesting site by a tissue barrier.
  • the animals are sacrificed at a predetermined time following administration of the library, the tissue is removed and the tissue is either processed immediately or frozen in liquid nitrogen (stored at -80°C) for processing at a later date.
  • the tissue samples are homogenised in PBS containing protease inhibitors and the homogenate is used to infect E.coli, thus permitting amplification of phages that were transported to the harvesting site tissue.
  • the phage display library is purified such as by either PEG precipitation or by sucrose or CsCl density centrifugation.
  • the phage display library is resuspended in PBS (or TBS) buffer and injected into the in vivo animal site, such as duodenum, jejunum, ileum, colon, ascending colon, transverse colon, descending colon, pelvic colon in the closed (or open) animal (rat, rabbit or other species) loop model.
  • the corresponding region of the GIT track exposed to or incubated with the phage display library can be recovered at the end of the experiments.
  • the washed tissue is homogenised in PBS containing protease inhibitors and the homogenate is used to infect E.coli, thus permitting amplification of phages which can bind tightly to the intestinal tissue.
  • the recovered intestinal tissue can be homogenised in suitable PBS buffers, washed repeatedly and the phage present in the final tissue homogenate can be amplified in E.coli. This latter approach also permits amplification of phages which either bind tightly to the intestinal tissue or which are internalized by the epithelial cells of the intestinal tissue
  • the purified phage display library (random or preselected) is diluted to 500 ⁇ l in PBS buffer and injected into the closed (or open) intestinal loop model (e.g., rat, rabbit or other species).
  • the closed (or open) intestinal loop model e.g., rat, rabbit or other species.
  • An aliquot of the withdrawn blood can be incubated with E. coli, followed by plating for phage plaques or for transduction units or for colonies where the phage codes for resistance to antibiotics such as tetracycline.
  • the remainder of the withdrawn blood sample (up to 150 ⁇ l) is incubated with 250 ⁇ l of E. coli and 5 ml of LB medium or other suitable growth medium.
  • coli cultures are incubated overnight by incubation at 37°C on a shaking platform. Blood samples taken at other time points (such as 15 min, 30 min, 45 min, 60 min up to 6 hours) are processed in a similar manner, permitting amplification of phages present in the portal or systemic circulation in E. coli. at these times.
  • the amplified phage is recovered by PEG precipitation and resuspended in PBS buffer or TBS buffer.
  • the titer of the amplified phage, before and after PEG precipitation is determined.
  • the amplified, PEG precipitated phage is diluted to a known phage titer (generally between 10 8 and 10 10 phage or plaque forming units per ml) and is injected into the GIT of the animal closed (or open) loop model.
  • a known phage titer generally between 10 8 and 10 10 phage or plaque forming units per ml
  • Blood samples are collected from portal and/or systemic ciruclation at various time points and the phage transported into the blood samples are amplified in E. coli as given above for the first cycle.
  • the phage are PEG precipitated, resuspended, titered, diluted and injected into the GIT of the animal closed (or open) loop model.
  • This procedure of phage injection followed by collection of portal and/or systemic blood samples and amplification of phage transported into these blood samples can be repeated, for example, up to 10 times, to permit the selection of phages which are preferentially transported from the GIT into the portal and/or systemic circulation.
  • the animal can be sacrificed and the harvesting site tissue, such as brain tissue, removed.
  • tissue such as brain tissue
  • the tissue can be processed immediately or frozen in liquid nitrogen (stored at -80°C) for processing at a later date.
  • serial dilutions such as neat, 10 "2 , 10 "4 , 10 "6 dilutions
  • E. coli E. coli.
  • tissue homgentate An aliquot (100 ⁇ l) of the tissue homgentate is added to 100 ⁇ l of E. coli K91 Kan starved bacteria, and incubated at 37°C for 10 min followed by addition of 5 ml of LB medium or other suitable growth medium. The E. coli cultures are incubated overnight at 37°C and serial dilutions of amplified phage are then titered in E. coli.
  • the recovered phage can be administered to other animals, harvesting site tissue can be collected, and the phage transported into the tissues can be amplified repeatedly, for example, up to 10 times, to permit the selection of phages which are preferentially transported from the GIT into the harvesting site tissue.
  • tissue culture cell monolayers with phage display populations In a laminar flow cabinet, 100 ⁇ l of phage solution was mixed with 900 ⁇ l of growth medium without antibiotic (the complete recommended medium for each cell line but with no antibiotics added) in a microfuge tube. The experiment was carried out in duplicate and included a control treatment containing no phage. The T ⁇ R was measured for each snapwell, noting the age of the cells and the passage number. Only intact monolayers of recommended age were used which had expected T ⁇ R. The basolateral medium was replaced in the snapwells with medium without antibiotic and the apical medium was removed. The phage solutions and control solutions were added to the apical side of the cells and the snapwell cultures were incubated as normal.
  • the medium was removed from the basolateral side and stored in a sterile 2 ml screwcap tube at 4°C. At each time that the basolateral medium is removed, the medium was replaced with fresh medium without antibiotic. When the experiments are finished, the TER was measured and the monolayers were treated with Vircon disinfectant as per normal.
  • Phage expressing presumed GIT binding peptide inserts were assayed by ELISA on fixed Caco-2 cells as follows. Cells were plated at 1 x 10 5 cells/well on 100 ⁇ l culture media and incubated at 30°C in 5% CO 2 overnight. 100 ⁇ l 25% formaldehyde was added to each well for 15 minutes. Contents of the wells were removed by inverting the plate. The plate was then washed 3 times with DPBS. 0.1%> phenylhydrazine DPBS solution was added to each well and incubated for 1 hr at 37°C. The plate was inverted and washed 3 times. The plate was blocked with 0.5%) BBA-DPBS for 1 hr at room temperature.
  • the plate was inverted and washed 3 times with 1% BPT. Phage diluted with 1% BPT was added to wells containing fixed cells. Wells without phage added were used to determine background binding of the HRP conjugate. The plates were incubated 2-3 hours on a rotor at room temperature. Plates were washed as before. Plates were incubated with dilute anti M13-HRP antibody in 1% BPF for 1 hour at room temperature. Following washing, TMB substrate was added and absorbance of the plates were read at 650 nm.. Nanoparticles were added to the cells at lOmg/ml in lOO ⁇ l 1%>BSA-PBS (no
  • Tween80 is used in this assay) and 2-fold serially-diluted.
  • the 96-well plates were incubated for lh at room temperature.
  • the plates were washed 5 times with 1% BSA- PBS and lOO ⁇ l of anti-dansyl antibody (Cytogen DB3-226.3; 0.5 ⁇ g/ml; batch May 1997) was added per well and the plates incubated lh at room temperature.
  • the wells were washed 5 times with 1% BSA-PBS and lOO ⁇ l of goat anti-mouse ⁇ :HRP antibody (Southern Biotechnology CN. 1060-05; 1 :10,000) was added per well and the plates incubated lh at room temperature.
  • lOO ⁇ l of TMB peroxidase substrate KPL CN. 50-76-00
  • Binding of nanoparticles coated with targeting peptides to fixed Caco-2 cells was investigated using an ELIS A assay based on reaction of antibody with the dansyl moiety present on the peptides.
  • Confluent Caco-2 monolayers grown in 96-well plates (p38) were fixed and treated with 0.1% phenylhydrazine before blocking with 0.1% BSA in PBS.
  • Control and dansyl peptide-coated nanoparticles were resuspended in sterile water at lOmg/ml and stirred with a magnet for lh at room temperature.
  • Formulations General Method for Preparation of Coacervated Particles.
  • Solid particles containing a Therapeutic as defined herein are prepared using a coacervation method.
  • the particles have a particle size of between about lOnm and 500 ⁇ m, most preferably 50 to 800 nm.
  • the particles contain targeting ligands which are incorporated into the particles using a number of methods.
  • particles are formed using the following general method:
  • the mixture (A) is stirred under low shear conditions at 10- 2000 rpm, preferably 100-600 ⁇ m.
  • the pH and/or ionic strength of this solution may be modified using salts, buffers or other modifying agents.
  • the viscosity of this solution may be modified using polymers, salts, or other viscosity enhancing or modifying agents.
  • a polymer preferably poly(lacide-co-glycolide) (hereinafter PLGA), polylactide, polyglycolide or a combination thereof or in any enantiomeric form or a covalent conjugate of the these polymers with a targeting ligand is dissolved in water miscible organic solvents to form organic phase (B).
  • PLGA poly(lacide-co-glycolide)
  • B organic phase
  • Most preferably a combination of acetone and ethanol is used in a range of ratios from 0:100 acetone: ethanol to 100:
  • Additional polymer(s), peptide(s) sugars, salts, natural/biological polymers or other agents may also be added to the organic phase (B) to modify the physical and chemical properties of the resultant particle product.
  • a drug or bioactive substance may be introduced into either the aqueous phase (A) or the organic phase (B).
  • a targeting ligand may also be introduced into either the aqueous phase (A) or the organic phase (B) at this point.
  • the organic phase (B) is added into the stirred aqueous phase (A) at a continuous rate.
  • the solvent is evaporated, preferably by a rise in temperature over ambient and/or the use of a vacuum pump.
  • the particles are now present as a suspension (C).
  • a targeting ligand may be introduced into the stirred suspension at this point.
  • a secondary layer of polymer(s), peptide(s) sugars, salts, natural/biological polymers or other agents may be deposited on to the pre-formed particulate core by any suitable method at this stage.
  • the particles (D) are then separated from the suspension (C) using standard colloidal separation techniques, preferably by centrifugation at high 'g' force, filtration, gel permeation chromatography, affinity chromatography or charge separation techniques. The supernatant is discarded and the particles (D) re-suspended in a washing solution (E) preferably water, salt solution, buffer or organic solvent(s).
  • a washing solution preferably water, salt solution, buffer or organic solvent(s).
  • the particles (D) are separated from the washing liquid in a similar manner to previously described and re-washed, commonly twice.
  • a targeting ligand may be dissolved in washing solution (E) at the final washing stage and may be used to wash the particles (D).
  • the particles may then be dried. Particles may then be further processed for example, tabletted, encapsulated or spray dried.
  • the release profile of the particles formed above may be varied from immediate to controlled or delayed release dependent upon the formulation used and/or desired.
  • Drug loading may be in the range 0-90% w/w.
  • Targeting ligand loading may be in the range 0-90% w/w.
  • the surface active agent of aqueous solution (A) in the general method above includes agents that may be referred to as emulsifmg agents, detergents, solubilizing agents, wetting agents, foaming and antifoaming agents, flocculants and defloculant.
  • anionic surface active agents such as sodium dodecanoate, sodium dodecyl(lauryl)sulphate, sodium dioctyl sulphosuccinate, cetostearyl alcohol, stearic acid and its salts such as magnesium stearate and sodium stearate, sodium dodecyl benzene sulphonate, sodium cholate triethanolamine; cationic surface active agents such as hexadecyl trimethyl ammonium bromide(cetrimide), dodecyl pyridinium iodide, dodecyl pyridinium chloride; non-ionic suface active agents such as hexaoxyethylene monohexadecyl ether, polysorbates(T weens), sorbitan esters(Spans), Macrogol ethers, Poloxalkols (Poloxamers),PVA, PVP, glycols and glycerol esters, fatty alcohol poly glycol ethers, dextrans
  • the organic phase (B) polymer of the general method given above may be soluble, permeable, impermeable, biodegradable or gastroretentive.
  • the polymer may consist of a mixture of polymer or copolymers and may be a natural or synthetic polymer.
  • biodegradable polymers include without limitation polyglycolides; polylactides; poly(lactide-co-glycolides), including DL, L and D forms; copolyoxalates; polycaprolactone; polyesteramides; polyorthoesters; polyanhydrides; polyalkylcyanoacrylates; polyhydroxybutyrates; polyurethanes; albumin; casein; citosan derivatives; gelatin; acacia; celluloses; polysaccharides; alginic acid; polypeptides; and the like, copolymers thereof, mixtures thereof and stereoisomers thereof.
  • Representative synthetic polymers include alkyl celluloses; hydroxalkyl celluloses; cellulose ethers; cellulose esters; nitro celluloses; polymers of acrylic and methacrylic acids and esters thereof; dextrans; polyamides; polycarbonates; polyalkylenes; polyalkylene glycols; polyalkylene oxides; polyalkylene terephthalates; polyvinyl alcohols; polyvinyl ethers; polyvinyl esters; polyvinyl halides; poyvinylpyrrolidone; polysiloxanes and polyurethanes and copolymers thereof.
  • the 5% w/v PVA solution is prepared by heating water until near boiling point, adding PVA and stirring until cool.
  • the organic phase is prepared by adding acetone, 45mls, and ethanol, 5mls, together.
  • the polymer solution is prepared by adding RG504H, 2g, to the organic phase and stirring until dissolved.
  • the IKA reactor vessel is set up, all seals greased and the temperature is set at 25°C.
  • the PVA solution, 400mls is added into the reactor vessel and stirred at 400 ⁇ m.
  • Bovine insulin lOOmg is added into the stirring PVA solution. Using clean tubing and a green needle the polymer solution, is slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent is allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 ⁇ m.
  • the suspension is centrifuged in a Beckman Ultracentrifuge with swing-out rotor at 12,500 ⁇ m and 4°C. The supernatant is decanted and discarded and the "cake" of particles is broken up. dH 2 O (200mls) is added to wash the particles. The centrifugation and washing steps were repeated twice.
  • the peptide solution (peptide 41.1 lOmg) in 50ml dH 2 O is prepared and added to the particles for a final washing stage.
  • the suspended particles were centrifuged as before.
  • the supernatant liquid is decanted off, the 'cake' broken up and the particles were dried in the vacuum oven.
  • the particles were ground, placed in a securitainer and sent for analysis.
  • Formulation 2 Peptide added at the beginning of manufacture
  • Product Bovine Insulin loaded nanoparticles
  • Bovine Insulin (Lot no. 65H0640) lOOmg
  • the 5% w/v PVA solution is prepared by heating water until near boiling point, adding PVA and stirring until cool.
  • the organic phase is prepared by adding acetone, 45mls, and ethanol, 5mls, together.
  • the polymer solution is prepared by adding RG504H, 2g, to the organic phase prepared in step above and stirring until dissolved.
  • the IKA reactor vessel is set up, all seals greased and the temperature is set at 25°C.
  • the PVA solution, 400mls, is added into the reactor vessel and stirred at
  • Bovine insulin lOOmg is added into the stirring PVA solution.
  • PAX 2 (ZELAN018U lOmg) is added to the stirring PVA solution.
  • the polymer solution (step 3), is slowly dripped in the stirring PVA solution with the peristaltic pump set at 40.
  • the solvent is allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 ⁇ m.
  • the suspension is centrifuged in a Beckman Ultracentrifuge with swing-out rotor at 12,500 ⁇ m and 4°C. The supernatant is decanted and discarded.
  • the "cake" of particles is broken up and dH 2 O (200mls) is added to wash the particles. The centrifugation and washing steps were repeated twice.
  • the 'cake' is broken up and the particles were dried in the vacuum oven.
  • the particles were ground, placed in a securitainer and sent for analysis.
  • Aim To prepare a lg batch of insulin loaded nanoparticles at a theoretical loading of 50mg/g and with the peptide 41.1 added 1 hour before centrifugation.
  • Bovine Insulin (Lot no. . 65H0640) 50mg
  • the 5% w/v PVA solution is prepared by heating water until near boiling point, adding PVA and stirring until cool.
  • the organic phase is prepared by adding acetone, 22.5mls, and ethanol, 2.5mls, together.
  • the polymer solution is prepared by adding RG504H, lg, to the organic phase prepared above and stirring until dissolved.
  • the IKA reactor vessel is set up, all seals greased and the temperature is set at 25°C.
  • the PVA solution, 200mls, is added into the reactor vessel and stirred at 400 ⁇ m.
  • Bovine insulin 50mg is added into the stirring PVA solution. Using clean tubing and a green needle the polymer solution, is slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent is allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 ⁇ m.
  • Peptide 41.1 is added to the stirring particle suspension. After 1 hr the suspension is centrifuged in a Beckman Ultracentrifuge with swing-out rotor at 12,500 ⁇ m and 4°C. The supernatant is decanted and discarded. The "cake" of particles is broken up and dH 2 O (200mls) is added to wash the particles. The centrifugation and washing steps are repeated twice.
  • the 'cake' is broken up and the particles were dried in the vacuum oven.
  • the particles were ground, placed in a securitainer and sent for analysis.
  • Aim To prepare a 3g batch of leuprolide-acetate loaded nanoparticles at a theoretical loading of 20mg/g and with the peptide 41.1 added.
  • Leuprolide acetate (Lot no. VI 4094) 60mg
  • Peptide P31 (Zelan024) 15mg/50ml dH 2 O
  • the PVA solution is prepared and the organic phase is prepared by adding acetone. 67.5mls, and ethanol, 7.5mls, together.
  • the polymer solution is prepared by adding RG504H, 3g, to the organic phase prepared above and stirring until dissolved.
  • the IKA reactor vessel is set up, all seals greased and the temperature is set at 25°C.
  • the PVA solution 600mls, is added into the reactor vessel and stirred at 400 ⁇ m.
  • Leuprolide acetate 60mg is added into the stirring PVA solution. Using clean tubing and a green needle the polymer solution, is slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent is allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 ⁇ m. The suspension is centrifuged in a Beckman Ultracentrifuge with swing-out rotor at 15 ⁇ m and 4°C. The supernatant is decanted and retained for analysis.
  • the "cake” of particles is broken up and dH 2 O 200mls) is added to wash the particles. The centrifugation and washing steps were repeated twice.
  • the peptide solution (peptide 41.1, 15mg) in 50ml dH 2 O is prepared and added to the particles for a final washing stage.
  • the suspended particles were centrifuged as before.
  • the supernatant liquid is decanted off and the particles were dried in the vacuum oven.
  • the particles were ground, placed in a securitainer and sent for analysis.
  • Aim To prepare a 3g batch of insulin loaded nanoparticles at a theoretical loading of 50mg/g and with the polymer-peptide conjugate PLGA-peptide 41.1 added.
  • the 5% w/v PVA solution is prepared by heating water until near boiling point, adding PVA and stirring until cool.
  • the organic phase is prepared by adding acetone, 67.5mls, and ethanol, 7.5mls, together.
  • the polymer solution is prepared by adding RG504H, and the polymer -peptide conjugate, to the organic phase prepared in step 2 and stirring until dissolved.
  • the IKA reactor vessel is set up, all seals greased and the temperature is set at 25°C.
  • the PVA solution, 400mls, is added into the reactor vessel and stirred at 400 ⁇ m.
  • Bovine insulin lOOmg is added into the stirring PVA solution. Using clean tubing and a green needle the polymer solution, (step 3), is slowly dripped in the stirring PVA solution with the peristaltic pump set at 40. The solvent is allowed to evaporate by opening the ports and allowing the dispersion to stir overnight at 400 ⁇ m.
  • the suspension is centrifuged in a Beckman Ultracentrifuge with swing-out rotor at 12,500 ⁇ m and 4°C. The supernatant is decanted and discarded. The "cake" of particles is broken up and dH 2 O (200mls) is added to wash the particles. The centrifugation washing step is repeated twice.
  • the 'cake' is broken up and the particles were dried in the vacuum oven.
  • the particles were ground, placed in a securitainer and sent for analysis.
  • phage from random phage display libraries as well as control phage were injected into the lumen of the rat gastro-intestinal tract (in situ rat closed loop model). Blood was collected over time from either the systemic circulation or portal circulation and the number of phage which were transported to the circulation was determined by titering blood samples in E. coli. At the conclusion of the collection of either systemic (300 min) or portal blood (60 min), the animals were sacrificed and brain, heart, kidney, spleen, ileum, duodenum, liver and pancreas tissue samples were removed, frozen in liquid nitrogen and stored at -80°C.
  • the phage display libraries used in this study were D38 and DC43 in which gene III codes for random 38-mer and 43-mer peptides, respectively.
  • As a negative control the identical phage M13mpl8, in which gene III does not code for a "random" peptide sequence, was used.
  • Both the library phages D38 and DC43 were prepared from E.coli, mixed together, dialyzed against PBS, precipitated using PEG/NaCl and were resuspended in PBS buffer.
  • the M13mpl8 control was processed in a similar manner. The titer of each phage sample was determined and the phage samples were diluted in PBS to approximately the same titers prior to injection into the rat closed loop model.
  • the estimated number of transported phage has been adjusted to account for differences in volume injected into each animal (using 0.5 ml as the standard volume).
  • animals Rl, R2 and R3 received the control phage M13mpl 8 and animals R4, R5, R6 and R7 received the test phage D38 / DC43 mix.
  • animals R8, R9 and R10 received the control phage M13mpl8 and animals Rl 1, R12, R13 and R14 received the test phage D38 / DC43 mix.
  • Animal R15* received the combined phage samples from animals R4-R7 (see Table 8) which were sampled from the systemic circulation on day one, followed by amplifiction in E. coli, PEG precipitation and resuspension in PBS. On subsequent analysis, the titer of this phage was found to 100 times greater than the other phage samples used for animals R8 - R14. Thus, the date presented for animal R15 in Table 9 is adjusted down.
  • phage was removed from the "amplified" supernatants obtained from test animals #R4-R7 (samples from each time point were used), combined and was PEG-precipitated for two hours.
  • the precipitated phage was resuspended in PBS buffer and was injected into closed loop model of animal #R15, followed by portal sampling.
  • the number of phage transported from the closed loop model into the systemic circulation is presented in Table 2.
  • the number of phage transported from the closed loop model into the portal circulation is presented in Table 3. These numbers are corrected for phage input difference and for volume input differences.
  • more phage are present in the portal samples than in the systemic samples, indicative of either hepatic or RES clearance and/or phage instability in the systemic circulation.
  • the uptake of phage from the GIT into the portal circulation is quite rapid, with substantial number of phages detected within 15 minutes.
  • the results from the portal sampling experiments would also indicate that the kinetics of uptake of phage from the D38 / DC43 libraries is quicker than that of the control phage.
  • % of the phage transported into the titered blood sample within the limited time frame (30, 45 and 15 mins, respectively) is estimated as 0.13%, 1.1% and 0.013%, respectively.
  • Animal R15* received the combined phage samples from animals R4-R7 (see Table 8) which were sampled from the systemic circulation on day one, followed by PEG precipitation and resuspension in PBS. On subsequent analysis, the titer of this phage was found to be 100 times greater than the other phage samples used for animals R8 - R14. Thus, the date presented for animal R15* in Table 9 is adjusted down.
  • Phage from the D38/DC43 libraries which appeared in the systemic circulation of different animals (R4-R7) were pooled, amplified in E.coli, precipitated, and re-applied to the lumen of the GIT, followed by collection in the portal circulation and titering in E.coli. These selected phage were also transported from the lumen of the GIT into the portal circulation.
  • This in situ loop model may represent an attractive screening model in which to identify peptide sequences which facilitate transport of phage and particles from the GIT into the circulation.
  • the frozen brain tissues for all the animals were thawed, cut into small pieces, resuspended in 5 ml of sterile PBS containing a cocktail of protease inhibitors (Boehringer) and homogenized in an Ultrathorex homogenizer. Serial dilutions (neat and 10 "2 , 10 "4 , 10 '6 dilutions) were titered in E. coli. K91 Kan starved bacteria.
  • tissue homogenate samples obtained from animals R4-R7 (sacrificed after systemic sampling) prior to amplification but phage were detected after amplification. Phage were detected prior to amplification in tissue honogenate samples obtained from animals Rl l-Rl 4.
  • phage from animals Rl 1 through R14 were pooled at a concentration of 5x10" pfu/ml (in PBS). These phage were amplified as above, plated and the recombinant phage were picked and purified. Sequencing templates were prepared using the QIAprep Spin Ml 3 columns and were sequenced using the primer S ⁇ Q. NO. ID: 17 and Sequenase Version 2.0 DNA sequencing kit. Eight DNA sequences (SEQ. NOS. ID: 1, 3, 5, 7, 9, 11, 13, 15) with corresponding peptide sequences SEQ. NOS. ID: 2, 4, 6, 8, 10, 12, 14, 16 were discovered.
  • peptide sequences which were isolated because of their ability to transport phage (particles) from the GI tract to the brain, are capable of facilitating the transport of an active agent, such as a micro- or nanoencapsulated active agent, through a human or animal tissue.
  • an active agent such as a micro- or nanoencapsulated active agent
  • Fig. 1 shows the binding profiles of brain selected phage to Caco-2 fixed cells.
  • Fig. 1 A Caco-2 P34, read at 30 min;
  • Fig. A(b) Caco-2 P36, read at 20 min; and
  • Fig. A(c) Caco-2 P34, read at 10 min.
  • the neat sample corresponds to 10 10 phage/well;
  • Fig. 2 shows the uptake and transport of brain selected phage 41.1 and 1.4 across Caco-2 cell monolayers.
  • Fig. 1 shows the binding profiles of brain selected phage to Caco-2 fixed cells.
  • Fig. 1 A Caco-2 P34, read at 30 min
  • Fig. A(b) Caco-2 P36, read at 20 min
  • Fig. A(c) Caco-2 P34, read at 10 min.
  • the neat sample corresponds to 10 10 phage/well
  • Fig. 2 shows the uptake and transport of brain selected phage 41.1 and 1.4 across Caco-2 cell monolayers.
  • FIG. 2A shows the titer of phage eluted by acid treatment of cell monolayers after 2hrs incubtion at 37°C
  • Fig. 2B shows the titer of cell-associated phage detected in the cell lysate after 2hrs incubation at 37°C
  • Fig. 2C shows the titer of phage from the basolateral medium after 6 hrs incubation at 37°C of Caco-2 cells with phage suspension on the apical side.
  • Fig. 2A shows the titer of phage eluted by acid treatment of cell monolayers after 2hrs incubtion at 37°C
  • Fig. 2B shows the titer of cell-associated phage detected in the cell lysate after 2hrs incubation at 37°C
  • Fig. 2C shows the titer of phage from the basolateral medium after 6 hrs incubation at 37°C of Caco-2 cells with phage suspension on the ap
  • the amino acid sequences and isoelectric points (pi) associated with various peptides identified from phage transported from the rat duodenal lumen to the brain are given in Table 4.
  • the first amino acid of each sequence can only be either arginine (R) or serine (S) because the first two nucleotides of the coding codon (AGX) belong to the restriction site Xhol (C TCG AG) used for the cloning of random peptides in the phage DNA. If the X base is G or A the coded amino acid is arginine; if the X base is C or T the coded amino acid is serine.
  • the sequences in bold, i.e. GCG (DC43 library) and A (D38 library), are conserved sequences used for the construction of the libraries (McConnell et al; Molecular Diversity 1 :165-176 (1995)).
  • Table 5 itemizes the charge of the peptides at different pH as analyzed by PCGENE software.
  • Figs. 3 and 4 show homologies of the brain-homing transport-binding peptides to known proteins.
  • the first ten homologies obtained from a Blast search against the non-redundant SwissProt database for each peptide are given in Fig. 3.
  • Particularly interesting homologies for peptides 41.1, 37.1 and 1.4 are reported in Fig. 4.
  • Of particular interest is the homology of the peptide 41.1 with human, rat and murine dynamin II protein shown in Fig. 4.
  • peptide 41.1 and a fragment of human dynamin II homologous to it have been synthesized and a dansyl group has been added at the N-terminal of the sequence in order to enable the detection of the peptide with anti-dansyl antibody. Additionally, fragments of peptide 41.2 were synthesized as shown in Table 7.
  • Example 5 Binding of Synthetic Peptides and Peptide-Coated Particles to S100 and P100 fraction derived from Caco-2 cells
  • Caco-2 cell membrane (P100) and cytosolic (SI 00) fractions were prepared using a modification of the method described in Kinsella, B. T., O'Mahony, D. J. & G. A. Fitzgerald, 1994, J. Biol. Chem. 269(47): 29914-29919.
  • Confluent Caco-2 cell monolayers grown in 75 cm 2 flasks for up to 1 week at 37°C and 5% CO 2 ) were washed twice in Dulbecco's PBS (DPBS) and the cells were harvested by centrifugation at 1000 ⁇ m after treatment with 10 mM EDTA-DPBS.
  • the cells were washed 3 times in DPBS and the final cell pellet was resuspended in 3 volumes of ice cold HED buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulphonyl fluoride (PMSF)).
  • the cells were allowed to swell for 5 min on ice prior to homogenisation for 30 sec.
  • the homogenates were centrifuged at 40,000 ⁇ m for 45 min at 4°C.
  • the supernatant (SI 00) was removed and the pellet (P100) was resuspended in HEDG buffer (20 mM HEPES (pH 7.67), 1 mM EGTA, 0.5 mM dithiothreitol, 100 mM NaCl, 10% glycerol, 1 mM PMSF). Protein concentrations were determined using the Bradford assay (Bradford, M. M., 1976, Anal. Biochem. 72: 248-254). Binding of peptide and/or peptide-coated PLGA particles to membrane (PI 00) and cytosolic (SI 00) fractions was assessed by detection of the dansyl moiety inco ⁇ orated in the peptide.
  • Costar ninety six well ELISA plates were coated with SI 00 and PI 00 fractions (100 ⁇ g/ml in 0.05 M NaHCO 3 ) overnight at 4°C.
  • the plates were blocked with 0.5% bovine serum albumin in DPBS for 1 h at room temperature and washed 3 times in 1% BSA-DPBS.
  • Peptide-coated particles or peptides were dispersed in the same buffer and added to the plates at concentrations in the range 0.0325 - 0.5 mg/well. After 1 h at room temperature the plates were washed 5 times in 1% BSA-DPBS and 100 ⁇ l of anti-dansyl antibody (Cytogen DB3-226.3; 0.5 ⁇ g/ml) was added per well.
  • the plates were incubated for 1 h at room temperature.
  • the wells were washed 3 times in 1% BSA-DPBS and 100 ⁇ l of goat anti-mouse IgG:HRP antibody (Southern Biotechnology 1060-05; 1 :10,000) was added per well.
  • the plates were incubated for 1 h at room temperature.
  • Fig. 5 shows the binding of peptide 41.1 and dynamin 41.2 to PI 00 (panel A) and SI 00 (panel B) Caco-2 fractions.
  • the subcellular fractions PI 00 and SI 00 were used at a concentration of 50 ⁇ g/ml and the background has been subtracted.
  • the synthetic peptides were evaluated in ELISA assays for binding to membrane PI 00 and cytosolic SI 00 fractions isolated from Caco-2 cells.
  • the samples consisted of peptides: (1) scrambled PAX2-coated nanoparticles, (2) HAX42-coated nanoparticles, (3) peptide 41.1 -coated particles, and (4) dynamin 41.2-coated particles.
  • HAX42 is the dansylated peptide:
  • HAX42 positive control
  • Scrambled PAX2 negative control
  • HAX42 is a randomly chosen dansylated peptide having the following structure:
  • the binding of peptide 41.1 to both PI 00 and SI 00 Caco-2 fraction is comparable to the binding of GIT targeting peptide, HAX42. Saturation of peptide binding was observed between 2 and 10 ⁇ g/ml for peptide 41.1. No binding was observed for the dynamin human homologous peptide 41.2. Binding of scrambled P AX2 negative control peptide was observed at the highest concentration of peptide used (1 ⁇ g/ml).
  • Fig. 6 shows that at a particle concentration of 62.5 ⁇ g/well, test peptide 41.1 -coated particles (TEXP2041 and dynamin 41.2-coated particles (TEXP2042) exhibited greater binding to both PI 00 (panel A) and SI 00 (panel A) fractions than the scrambled PAX2 coated control particles or particles with no peptide coating (TEXP 939).
  • TEXP2041 there was a concentration effect with increasing P100 and SI 00 concentration, while there was not such a trend with the test particle TEXP2042.
  • test solution was injected directly into the ileum.
  • Wistar rats 300-35 Og were fasted for 4 hours and anaesthetized by IM administration 15 to 20 minutes prior to administration of the test solution with a solution of ketamine [0.525 ml of ketamine (100 mg/ml) and 0.875 ml of ACP (2mg/ml)].
  • the rats were then injected with a test solution (injection volume: 1.5ml PBS) intra-duodenally at 2-3 cm below the pyloris.
  • the test solution contained polylactide-co-glycolide (PLGA) particles manufactured according to the coacervation formulation 1 given above.
  • PLGA polylactide-co-glycolide
  • Insulin fast-acting bovine; 28.1 iu/mg was inco ⁇ orated in the particles at 300iu insulin (-210 mg particles).
  • Blood glucose values for the rats were measured using a Glucometer (Bayer; 0.1 to 33.3 m mol/L); plasma insulin values were measured using a Phadeseph RIA Kit (Upjohn Pharmacia; 3 to 240 ⁇ U/ml-assayed in duplicate). Systemic and portal blood was sampled.
  • Insulin loaded, peptide coated PLGA particles (TEXP2041 and TEXP2042) have been analyzed in vivo in the rat duodenal open loop model and the profiles of insulin delivery to the systemic circulation is reported in Fig. 7. While the profile of insulin concentration for TEXP2042 particles (dynamin 41.2) is higher than uncoated particles (to be noticed that the value at 3hrs need to be adjusted for a sample missing), the insulin detected in rats treated with TEXP2041 particles (peptide 41.1) is unexpectedly low.
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Abstract

L'invention concerne des agents de ciblage permettant ou facilitant le transport d'un agent actif dans des tissus gastro-intestinaux humains ou animaux. Ces agents de ciblage sont des peptides, ou leurs dérivés (par exemple des fragments) et leur peptidomimétique, ainsi que les séquences codant ces peptides et leurs dérivés. L'utilisation de ces agents de ciblage facilite le transport d'agents actifs de la lumière du tractus gastro-intestinal à l'ensemble du système cardio-vasculaire, et/ou cible lesdits agents actifs vers ledit tractus gastro-intestinal. L'invention concerne également des procédés d'administration thérapeutiques ainsi que des compositions et des formulations pharmaceutiques à base des peptides agents de ciblage. L'agent actif est de préférence un médicament, ou une microparticule ou une nanoparticule renfermant un médicament.
EP98923466A 1997-05-15 1998-05-15 Peptides favorisant le transport d'un agent actif dans les tissus, compositions les renfermant et leurs procedes d'utilisation Withdrawn EP0981649A4 (fr)

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US857046 1986-04-29
US08/857,046 US6361938B1 (en) 1996-11-08 1997-05-15 Peptides which enhance transport across tissues and methods of identifying and using the same
PCT/US1998/010079 WO1998051825A1 (fr) 1997-05-15 1998-05-15 Peptides favorisant le transport d'un agent actif dans les tissus, compositions les renfermant et leurs procedes d'utilisation

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US6361938B1 (en) 2002-03-26
JP2002502368A (ja) 2002-01-22
CA2290071A1 (fr) 1998-11-19
WO1998051825A1 (fr) 1998-11-19
US7566766B2 (en) 2009-07-28
US20040023204A1 (en) 2004-02-05
AU7575698A (en) 1998-12-08
EP0981649A4 (fr) 2003-05-02

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